Packing cell system for eb virus vector

ABSTRACT

A packaging cell is constructed by preparing a packaging cell carrying an EB virus gene lacking a packaging signal but not carrying a wild type EB virus gene having the packaging signal, and introducing an amplicon plasmid having the packaging signal but having no viral replication ability into the cell. By inducing lytic infection of the packaging cell introduced with an amplicon plasmid, an EB virus vector without viral replication ability is produced.

TECHNICAL FIELD

The present invention relates to a method for producing an EB virusvector, and a packaging cell system used for the method.

BACKGROUND ART

In transferring genes into cells, a substance which plays a role intransferring genes into a target cell is called “vector”. From theviewpoint of safety, a virus-derived vector from which genes derivedfrom the original wild type virus have better to be removed as much aspossible. Epstein-Barr virus (hereinafter, referred to EB virus), whichis one of the herpes viruses, has an ability to infect human Blymphocytes and promotes their proliferation. When compared withcurrently used vectors based on other viruses, the EB virus vector hasmany advantages of, for example, 1) being to be maintained stably in atransfected cell for a long period of time; 2) ability to incorporatelarge foreign genes of over 100 kb in size; 3) ability to introduce agene efficiently into resting B lymphocytes, particularly, primary Blymphocytes; 4) ability to introduce and express a gene into Blymphocytes which are known to work as antigen presenting cells; 5)ability to propagate the B lymphocytes, into which a gene is introduced,in vitro; 6) ability to facilitate to prepare a vector efficiently, whena system of preparing a recombinant virus in E. coli is used; and thelike.

As cell strains producing EB virus, there are known Akata cell, B95-8cell, P3HR-1 cell and the like. Among them, Akata cell has about 20copies of circular EB virus genomes in the nucleus, and can induceinfectious EB virus production by adding an anti-human immunoglobulinantibody into a liquid culture medium. Therefore, Akata cell is a cellstrain suitable to produce EB virus in large amount (Takada K, Int JCancer 33, 27-32 (1984); Takada K and Ono Y, J Virol 63, 445-449(1989)). A clone in which an EB virus genome was dropped off wasobtained by subculture of Akata cell for a long period (hereinafter,referred to as “EB virus-negative Akata cell”). The EB virus-negativeAkata cell can be re-infected with EB virus and repeatedly express anability to produce the virus by treatment with an anti-humanimmunoglobulin antibody (Japanese Patent Laid-Open No. Hei 7-115966). Inaddition, an Akata cell having only a recombinant EB virus genome can beproduced, by generating a homologous recombinant EB virus having a drugmarker gene in Akata cell, inducing EB virus production, andre-infecting an EB virus-negative Akata cell with the resulting virusand then selecting a cell with the drug. This cell has an ability toproduce a recombinant EB virus in large amount by treatment with ananti-human immunoglobulin antibody (Japanese Patent Laid-Open No. Hei.8-009971).

There is a report which mentions methods for integrating a circular EBvirus genome originated from EB virus B95-8 strain in an E. coliartificial chromosome and for multiplying the integrated form in E. coli(U.S. Pat. No. 6,291,246). By using this system, mutation can beintroduced efficiently even in a gene essential for replication andproliferation of EB virus by homologous recombination in E. coli. Aresearch group in Germany discloses a method to introduce an EB virusgenome, from which a packaging signal was eliminated in E. coli system,into a 293 cell originated from fetal kidney and to apply the cell as apackaging cell for producing EB virus vector (Delecluse H J, Pich D,Hilsendegen T, Baum C, Hammerschmidt W. Proc Natl Acad Sci USA 96,5188-5193 (1999)). In the method, EB virus vector production is inducedby extraneously and simultaneously introducing an EB virus early geneBZLF1 and amplicon plasmid into the 293 cell and allowing to expressthem. However, in this system, means for EB virus vector production arecomplicated, and additionally, the titer of the produced EB virus vectoris very low. Therefore, this system is not suitable for production of EBvirus vectors in large quantity.

A system for efficient introduction of a foreign gene into a cell byusing an EB virus envelope is reported (Delecluse H. J, Hammerschmidt W,J Clin Pathol: Mol Pathol 2000; 53: 270-279, and the like). A method forpackaging a gene vector DNA without contamination of any helper virus isalso reported (Japanese Patent Laid-Open No. Hei 11-221073).

An object of the present invention is to provide a system for efficientproduction of a large amount of EB virus vector lacking viralreplication ability.

DISCLOSURE OF THE INVENTION

A method for producing a packaging cell in order to construct a systemfor producing an EB virus vector lacking viral replication ability isprovided.

In the first embodiment, the method of the present invention comprisesthe steps of:

-   introducing a gene fragment for homologous recombination lacking a    packaging signal into an EB virus-positive Akata cell, thereby    deleting a packaging signal of EB virus by homologous recombination,    and-   cloning a packaging cell carrying an EB virus genome lacking a    packaging signal, but not carrying a wild type EB virus genome    having a packaging signal.

In the second embodiment, the method of the present invention comprisesthe steps of:

-   preparing an EB virus genome lacking a packaging signal in the    bacterial E. coli cell, introducing the EB virus genome into an EB    virus-positive Akata cell, and cloning a packaging cell carrying an    EB virus genome lacking a packaging signal, but not carrying a wild    type EB virus genome having a packaging signal.

In the third embodiment, the method of the present invention comprisesthe steps of:

-   preparing an EB virus genome lacking a packaging signal by the use    of E. coli, introducing the EB virus genome into an EB    virus-negative Akata cell expressing EBNA1, and-   cloning a packaging cell carrying an EB virus genome lacking a    packaging signal, but not carrying a wild type EB virus genome    having a packaging signal.

In another aspect, the present invention provides a method forconstructing an Akata packaging cell introduced with an ampliconplasmid, which is used for producing an EB virus vector lacking viralreplication ability. The method comprises the step for introducing anamplicon plasmid having a packaging signal, but lacking viralreplication ability, into a packaging cell obtained by any one of theabove methods.

In still another aspect, the present invention provides a method forproducing an EB virus vector lacking viral replication ability,comprising induction of lytic infection of an Akata packaging cellintroduced with an amplicon plasmid, which is produced by the abovemethod of the present invention, thereby allowing an EB virus vectorcovered with a virus envelope to be released.

In further still another aspect, the present invention provides a methodfor producing an immortalized B lymphocyte, comprising inducing lyticinfection of an Akata packaging cell introduced with an ampliconplasmid, which is produced by the above-mentioned method of the presentinvention, thereby allowing an EB virus vector covered with a virusenvelope to be released, and infecting a B lymphocyte with the resultingEB virus vector.

According to the present invention, it is possible to produce simply andefficiently an EB virus vector lacking viral replication ability throughinduction of lytic infection of a packaging cell introduced with anamplicon plasmid, which is prepared by introducing an amplicon plasmidinto the packaging cell.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the method for preparing a packaging cell of the presentinvention.

FIG. 2 shows production of an EB virus vector by the Akata packagingcell introduced with an amplicon plasmid.

FIG. 3 shows construction of a plasmid for cloning of a packaging signalgene (TR) and a targeting plasmid for disrupting the packaging signal.

FIG. 4 shows a position of primers used for confirmation of homologousrecombination of the gene.

FIG. 5 shows a position of primers used for gene amplification in orderto confirm dropping off of wild type EB virus.

FIG. 6 shows one of the examples for a construction of both TR deficientEB virus genome and amplicon plasmid which is present in the packagingcell introduced with an amplicon plasmid.

FIG. 7 shows production of EB virus vector by the packaging cellintroduced with an amplicon plasmid.

FIG. 8 shows generation of a virus incapable of replicating its ownviral genome, and production of the EB virus vector incapable ofreplicating its own viral genome using a packaging cell into which thevirus incapable of replicating its own viral genome is introduced byinfection.

FIG. 9 shows construction of a targeting plasmid for disruption anddeletion of BALF2 gene and a probe used for detecting viruses resultingfrom homologous recombination.

FIG. 10 shows positions of primers used for gene amplification in orderto confirm production of a virus lacking BALF2 gene by homologousrecombination.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method for producing a packaging cellused for preparing a system for producing an EB virus vector withoutviral replication ability.

“Packaging cell” is a cell having an EB virus genome which lacks itspackaging signal but is able to express viral proteins necessary forproduction of virus particles. The packaging signal is a cis-actingsequence necessary for each viral genome to be packaged in an EB virusparticle, and it is also referred as TR (Terminal Repeat). In thepresent specification, the term “lacking (or deletion)” includes both acase where the packaging signal is totally deleted and a case where thepackaging signal is partially deleted or mutated whereby a function as apackaging signal is lost.

“EB virus vector” means a virus particle covered with an EB virusenvelope, having an ability to infect human B lymphocytes. The EB virusvector may contain a foreign gene. “EB virus vector without viralreplication ability” means a vector which is able to infect a host celland replicate the genome in the cell, but unable to be released as anenveloped viral particle, when lytic infection is induced in theinfected cell.

The Akata cell used in the present invention is a known humanlymphocyte, has about 20 circular EB virus genomes in the nucleus, andis capable of producing a recombinant EB virus generated by a homologousrecombination method (Japanese Patent Laid Open No. Hei 8-009971). Inaddition, since Akata cell can be separated to a clone in which an EBvirus genome has been dropped off by passaging the cell for a longperiod (Japanese Patent Laid Open No. Hei 7-115966). Therefore, a cellclone carrying only a recombinant EB virus genome, wherein a wild typeEB virus genome has been dropped off, can be separated by the samemethod. Moreover, Akata cell can produce a large amount of EB virus bytreatment with an anti-human immunoglobulin antibody (Takada K, Int JCancer 33, 27-32 (1984); Takada K and Ono Y, J Virol 63, 445-449(1989)).

The first embodiment of the method of the present invention isschematically shown in FIG. 1. First, a packaging signal of an EB virusis deleted by homologous recombination in an EB virus-positive Akatacell. This step is performed by introducing a gene fragment forhomologous recombination, which has regions sandwiching the packagingsignal therebetween and lacks the packaging signal itself, into an EBvirus-positive Akata cell. The gene fragment for homologousrecombination has a length of preferably 30 kb or less, more preferably20 kb or less. Such a gene fragment can be constructed with the use ofthe E. coli system based on the known EB virus genomic sequence.Introduction of a gene can be carried out by electroporation method,calcium phosphate method, lipofection method, gene gun method and thelike. Next, an Akata cell clone having both wild type Akata EB virusgenomes and an Akata EB virus genome lacking a packaging signal isseparated by utilizing drug resistance. In FIG. 1, a neomycin resistantgene is used for selecting the recombinant virus(es). Any selectionmarkers having function in Akata cell may also be utilized. Next, theseparated cell clones are treated with hydroxyurea and separated againinto cell clones, thereby isolating a packaging cell in which a wildtype virus has been dropped off and carrying only a virus lacking thepackaging signal. Cloning can also be carried out by limiting dilutionafter the passage culture without using hydroxyurea.

In the second embodiment of the method of the present invention, thestep of deleting a packaging signal of an EB virus by homologousrecombination is carried out as follows: by incorporating an EB virusDNA into an E. coli artificial chromosome and introduce the DNA into E.coli, by preparing an EB virus genome having no packaging signalutilizing gene recombination in E. coli cell, and by introducing theresulting EB virus genome into an EB virus-positive Akata cell. It isbecause an EB virus-positive Akata cell exhibits higher efficiency offoreign gene introduction as compared with an EB virus-negative Akatacell, and can proliferate more stably.

Specifically, first, to prepare a gene fragment for homologousrecombination, an E. coli artificial chromosome (BAC) vector fragmentfrom pBeloBAC11 (manufactured by Genome Systems Inc.) is inserted into agene fragment from an EB virus genome. Then, the resulting gene fragmentis introduced into an Akata cell having a wild type circular EB virusgenome, and thereafter an Akata cell having a homologously recombined EBvirus is selected. Next, a plasmid containing both a BAC and an Akata EBvirus genome is obtained by transforming E. coli with a genomic DNAprepared from the above clone. If an E. coli gene recombination systemis used, deletion and mutation can be introduced at any sites in EBvirus genome on this plasmid. Homologous recombination can be takenplace in Akata cell by introducing BAC-EB virus DNA thus prepared intoAkata cell, wherein the BAC-EB virus DNA lacks a packaging signal.

In the third embodiment of the method of the present invention, the stepof deleting a packaging signal of EB virus by homologous recombinationis carried out: by incorporating EB viral DNA into an E. coli artificialchromosome and introduce the DNA into E. coli, by preparing an EB virusgenome lacking a packaging signal utilizing the gene recombinationsystem in E. coli cell, and by introducing the resulting EB virus genomeinto an EB virus-negative Akata cell expressing EBNA1. The Akata cellexpressing EBNA1 can be cloned from an EB virus-negative Akata celltransfected with a linear DNA in which an EBNA1 gene is ligated with asuitable antibiotic-resistant gene, followed by the selection using theantibiotic. It is thought that efficiency of episome formation is highin an Akata cell expressing EBNA1, since the EBNA1 protein is anessential protein for replication and maintenance of EB virus as anepisome in the nucleus.

The present invention also provides a packaging cell produced by theabove method. When lytic infection is induced in the Akata packagingcell introduced with an amplicon plasmid, which is prepared byintroducing a foreign gene containing a packaging signal (amplicon) intothe packaging cell of the present invention, only the amplicon DNA isproduced as an EB virus vector coated with a virus envelope by the helpof EB virus gene products provided from a TR(−) EB virus genome. Thatis, the packaging cell of the present invention has a helper functionfor producing an EB virus vector containing foreign gene(s) and havingno viral replication ability.

Namely, in another aspect, the present invention provides a method forproducing the Akata packaging cell introduced with an amplicon plasmidfor producing an EB virus vector without viral replication ability. Anoutline of this method is shown in FIG. 2. An Akata packaging cellcontaining an amplicon plasmid is cloned by introducing an ampliconplasmid into the packaging cell carrying only an EB virus genome lackinga packaging signal but not carrying a wild type EB virus genome withpackaging signal(s), and by selecting the cell with an appropriate drug.Introduction of an amplicon plasmid can be carried out byelectroporation, calcium phosphate method, lipofection method, gene gunmethod and the like.

“Amplicon plasmid” means a plasmid (circular DNA) containingvirus-derived genes, however the plasmid lacks the ability to produce avirus. An amplicon plasmid is a circular DNA containing a packagingsignal and a virus replication origin, and lacking one or more genesencoding a protein essential for virus replication. Preferably, anamplicon plasmid has selection marker genes. An amplicon plasmid isparticularly preferred to have a packaging signal, a replication originoriP during EB virus latent infection, a replication origin oriLytduring EB virus lytic infection, and EBNA1. Furthermore, when an EBvirus vector is used in order to introduce foreign genes, an ampliconplasmid has the additional foreign genes to be introduced.

In FIG. 2, since an amplicon plasmid has a puromycin resistant gene as aselection marker, puromycin was used for selection of the Akatapackaging cell introduced with an amplicon. In this case, any selectionmarkers which function in this cell may be utilized. It is preferred touse selection markers different from selection markers for generatingthe packaging cell.

To introduce an amplicon plasmid into a packaging cell, a method basedon infection with a virus of which genome is inreplicable may also beused. An outline of this method is shown in FIG. 8. A gene important forreplication of an EB virus genome is deleted by homologous recombinationin an EB virus-positive Akata cell. In FIG. 8, a hygromycin resistantgene is used to select a homologous recombinant virus. By treating anAkata cell clone having both wild type virus and isolated homologousrecombinant virus was treated with anti-human immunoglobulin, deficientEB virus gene products are supplied from wild type viruses, therebyproducing both a homologous recombinant virus and a wild type virus. Apackaging cell is infected with the virus liquid solution produced andthen cloned with a selection marker (neomycin) used for generating thepackaging cell as well as a selection marker (hygromycin) used forgenerating replication incompetent virus, thereby isolating the Akatapackaging cell containing only a TR(−) EB virus gene lacking a packagingsignal and an EB virus gene lacking genes important for replication ofthe viral genome. By treating the resulting Akata packaging cellcontaining as an amplicon an EB virus gene lacking a gene important fora viral genomic replication, with an anti-human immunoglobulin antibody,only an amplicon plasmid with an EB virus envelope is released as an EBvirus vector.

The present invention also provides an Akata packaging cell having anamplicon plasmid produced by the above method. The Akata packaging cellhaving an amplicon plasmid of the present invention has an ability toproduce an amplicon plasmid DNA coated with an EB virus envelope as anEB virus vector by the treatment of the cell with an anti-humanimmunoglobulin antibody. An EB virus genome is elongated and replicatedin a rolling circle-manner. When the packaging signal is deleted, the EBvirus genome cannot be excised. Thus, the EB virus genome is notincorporated into a virus particle. On the other hand, a gene of anamplicon plasmid is also elongated in a rolling circle-manner.Thereafter, an amplicon forms a concatemer and is cut at a portion of apackaging signal, followed by packaging at a size of about 170 kbcorresponding to the size of an EB virus genome (see, FIG. 7). Thus,only an amplicon plasmid covered with an EB virus-envelope is releasedas an EB virus vector. It is preferable that the Akata packaging cellcontaining an amplicon plasmid generated by the method of the presentinvention produces an EB virus vector without viral replication abilityat an efficiency of at most 1,000 times or more than that by theconventional method does. And the Akata packaging cell does not producewild type EB virus.

Namely, in another aspect, the present invention provides a method forproducing an EB virus vector without viral replication ability,comprising inducing lytic infection of an Akata packaging cellintroduced with an amplicon plasmid, which is produced by theabove-mentioned method of the present invention, thereby releasing an EBvirus vector covered with a virus envelope. Induction of lytic infectionof an Akata packaging cell introduced with an amplicon plasmid can becarried out easily by treatment of the cell with an anti-humanimmunoglobulin antibody.

The present invention also provides an EB virus vector produced by theabove-mentioned method. This EB virus vector contains an ampliconplasmid within an EB virus particle. This EB virus vector is able toinfect B-lymphocytes and immortalize them. However, the EB virus vectordoes not have an ability to replicate itself as a virus particle.Therefore, a human antibody can be produced easily, by infecting a Blymphocyte producing a desired antibody with an EB virus vector of thepresent invention, by immortalizing the B lymphocyte, and byproliferating the B lymphocyte in vitro. Namely, the present inventionalso provides a method for generating an immortalized B lymphocyte usingan EB virus vector of the present invention as well as an immortalized Blymphocyte produced by the method of the present invention. Moreover, byincorporating gene of interest into an amplicon plasmid, it becomespossible to introduce the gene into resting B-lymphocytes in peripheralblood, which has been difficult. By using a promoter capable ofexpressing a gene only in resting B-lymphocytes, there can be expectedB-lymphocyte-specific and high-level expression of the gene.

Entire subject matters of all patent and reference literatures citedexplicitly in the present specification are incorporated herein as partof the present specification. Entire subject matters described in thespecification and drawings of Japanese Patent Application No.2002-349467 that serve as the basis for claiming priority of the instantapplication are also incorporated herein as part of the presentspecification.

EXAMPLES

The present invention will be explained further in detail by thefollowing examples, but the present invention is not limited to theseexamples.

Example 1

In this example, a packaging signal of Akata EB virus was disruptedbased on the method shown in FIG. 1. First, an 8 kb Bam-Nhet region ofAkata EB virus was cloned into pUC119, to construct pUC119Nhet (FIG. 3a). The cloned Bam-Nhet region harboured only one packaging signal (TR).Next, plasmid pUCNhetneoRed for disruption of the gene in which TR hadbeen disrupted was constructed by inserting a neomycin resistant gene(neo^(r)) and a red dye gene (DsRed) to a region between BspHI and EcoRIrestriction enzyme site of pUC119Nhet (FIG. 3 b). This plasmid DNA wasintroduced into an EB virus-positive Akata cell by the electroporationmethod.

Subsequently, Akata cell was selected with neomycin and about 500resistant Akata cell clones were obtained. Then, Akata cell cloneshaving a DNA band of a size which appears in the case of homologousrecombination were screened by a DNA hybridization method. A DNAfragment digested with a restriction enzyme BamHI was reacted with aneomycin resistant gene as a probe, to thereby detect a predicted 11.6kb-band.

Next, a gene amplification method was performed in order to confirmwhether or not homologous recombination was taken place. ANC F1 and ANCR2 are primers designed at the external regions of the 5′ end and 3′ endof the recombination site, respectively. ANC R1 and ANC F2 are primersdesigned at the internal region of neo^(r). Approximate positions anddirections of respective primers are shown in FIG. 4. In the case wherehomologous recombination is taken place, a 5.3 kb DNA fragment isamplified by primer pairs of ANC F1 and ANC R1, and a 10.3 kb DNAfragment is amplified by primer pairs of ANC F2 and ANC R2. Whenhomologous recombination is not taken place, no fragment is amplified.With using this method, it was confirmed by this method that 12 cloneswere carrying a homologous recombinant EB virus.

Next, an Akata cell having only a TR(−) virus genome in which apackaging signal had been disrupted by homologous recombination wasisolated. A 119 cell which was a clone carrying a TR(−) EB virus genomewas cultured in a selection medium (50% EB virus-negative Akata cellculture supernatant, 0.35 mg/ml G418, 50 μM hydroxyurea) for 10 days.Then, hydroxyurea was removed from the culture and cells were furthercultivated for 10 days. The resulting culture was diluted so as to place0.5 cell per well of a 96-well plate and then cultured. For theproliferated clone, gene amplification was carried out using primerpairs designed at the external regions of TR, to isolate an Akata cellclone in which a band of wild type (2.0 kb) had been dropped off andonly a band (5.7 kb) of recombinant type was observed. Further, based onchange in a DNA restriction enzyme digestion pattern by a southern blotmethod, it was confirmed that a packaging cell having only an EB viruslacking a packaging signal was isolated.

Example 2

In this example, an amplicon plasmid was prepared. As an ampliconplasmid, it is necessary to have a construction having a packagingsignal (TR). Further, it is important for maintenance of the plasmid inthe cell to be introduced that the amplicon plasmid should haveconstructions such as, replication origin oriP during an EB virus latentinfection period and an EBNA1 gene which trans-activates oriP. Inaddition, in order to produce an EB virus vector from the packaging cellintroduced with an amplicon plasmid, by stimulation with an anti-humanimmunoglobulin antibody, it is necessary for the amplicon plasmid tohave a replication origin oriLyt during the induced EB virus lyticinfection period. FIG. 6 b shows the construct of the generated ampliconplasmid. The amplicon plasmid (pPsi) is a 21 kb and contains EBNA-1,oriP, TR, and oriLyt, as EBV genes, wherein a green fluorescent protein(GFP) gene used as a reporter and a puromycin resistant gene used forselection, by which an amplicon plasmid is maintained in a cell, areadditionally incorporated. Although a genome of a helper virus iselongated and amplified in a rolling circle-manner, the genome is notexcised because of the deficit of TR in its genome. Therefore, thehelper virus genome is not incorporated into a virus particle. On theother hand, a gene of an amplicon plasmid is also elongated in a rollingcircle-manner. Thereafter, an amplicon forms a concatamer. Theconcatamer is cleaved at the TR portion at a size of about 170 kbcorresponding to the size of an EB virus genome, followed by packaging(FIG. 7). That is, an EB virus containing an amplicon plasmid for itsgene is released.

Example 3

In this example, the constructed amplicon plasmid pPsi was transfectedinto a packaging cell by electroporation. Thereafter, selective culturewas carried out in the medium containing 0.35 μg/ml puromycin, 50% EBvirus-negative Akata cell culture supernatant, and 0.35 mg/ml G418. Theresulting 72 puromycin resistant clones were all GFP-positive. One ofthe clones was treated with an anti-human immunoglobulin antibody.Thereafter, it was confirmed that a capsid antigen (VCA), which is aconstituent protein of an EB virus, was expressed and that a wild typevirus was not present in its supernatant. Further, a recombinant EBvirus in the supernatant was infected to an EB virus-negative Daudicell. As the result, it was confirmed that less than 20% of the Daudicells were infected with a recombinant EB virus and expressed GFP.

Example 4

In this example, BALF2 gene encoding EB virus single-stranded DNAbinding protein was disrupted by deleting the gene from the Akata EBvirus genome through substitution with a hygromycin resistant gene basedon the method in FIG. 8. First, a plasmid pUC119 EcoD-JHyg for thedisruption of the BALF2 gene was prepared by cloning an 11,429 bpEcoRI-SalI fragment encompassing the Eco-Dhet and Eco-J regions of AkataEB virus genome into pUC119. From pUC119 EcoD-JHyg, a 2,500 bp Cla I-EcoRV region was eliminated and substituted with hygromycin resistant geneof 1,853 bp (FIG. 9 a). A 7,807 bp Sac I-Bgl II fragment was excisedfrom this plasmid DNA and transfected into an EB virus-positive Akatacell by electroporation.

Subsequently, an Akata cell clone having a DNA band of a size appearingin the case of occurrence of homologous recombination was screened bycarrying out hygromycin selection of Akata cell, and then carrying out aDNA hybridization method for about 1,000 resistant clones. A DNAfragment digested with a restriction enzyme NcoI was reacted with thehygromycin resistant gene shown in FIG. 9 b as a probe, to therebydetect a predicted 4.8 kb band on DNA separated from 5 Akata cellclones.

Next, whether or not homologous recombination was taken place wasexamined by a gene amplification method. F1 and R2 are primers designedat the each external region of 5′ end or 3′ end of the recombinationsite, respectively. HygF and HygR are primers designed at the internalregion of the hygromycin resistant gene, respectively. Approximatepositions and directions of respective primers are shown in FIG. 10. Inthe case where homologous recombination is taken place, a 4,112 bp DNAfragment is amplified by a primer pair of F1 and HygR, and a 4,534 bpDNA fragment is amplified by a primer pair of HygF and R2. In the casewhere homologous recombination is not taken place, no DNA fragment isamplified. It was confirmed that 2 clones carried a homologousrecombinant EB virus by this method.

Next, a clone carrying a homologous recombinant EB virus was treatedwith an anti-human immunoglobulin antibody, to produce a mixture ofvirus containing both recombinant type and wild type. The produced virusin the liquid solution was infected to the packaging cell prepared inExample 1, and the cell was subjected to cloning in the presence ofneomycin and hygromycin. Subsequently, an Akata packaging cellcontaining only an EB virus genome lacking BALF2 gene and a TR(−) EBvirus genome as a helper, and having no wild type virus genome wasseparated. This Akata packaging cell was treated with an anti-humanimmunoglobulin antibody, to recover an EB virus vector containing only aBALF2 gene-deficient EB virus. A half million umbilical cordblood-derived lymphocytes were infected with 10-fold serially dilutedBALF2 gene-deficient EB virus liquid solution, each in the volume of 100μl, and then cultured for 4 weeks. As a result, it was confirmed that anEB virus vector having about 10⁵ TD₅₀/ml transforming activity buthaving no proliferation ability was produced.

1. A method for producing a packaging cell for preparing a system forproducing an EB virus vector without viral replication ability,comprising the steps of: introducing a gene fragment for homologousrecombination lacking a packaging signal into Akata cell, therebydeleting packaging signals of EB virus by homologous recombination, andcloning a packaging cell carrying an EB virus genome lacking a packagingsignal, but not carrying a wild type EB virus genome having packagingsignals.
 2. A method for producing a packaging cell for preparing asystem for producing an EB virus vector without viral replicationability, comprising the steps of: preparing an EB virus genome lacking apackaging signal by the use of E. coli, introducing the EB virus genomeinto an EB virus-positive Akata cell, and cloning a packaging cellcarrying an EB virus genome lacking a packaging signal, but not carryinga wild type EB virus genome having packaging signals.
 3. A method forproducing a packaging cell for preparing a system for producing an EBvirus vector without viral replication ability, comprising the steps of:preparing an EB virus genome lacking a packaging signal by the use of E.coli, introducing the EB virus genome into an EB virus-negative Akatacell expressing EBNA1, and cloning a packaging cell carrying an EB virusgenome lacking a packaging signal, but not carrying a wild type EB virusgenome having packaging signals.
 4. A method for producing an Akatapackaging cell introduced with an amplicon plasmid, which is used forproducing an EB virus vector without viral replication ability,comprising the step of: introducing an amplicon plasmid having apackaging signal, but lacking viral replication ability into a packagingcell obtained by the method of any one of claims 1 to
 3. 5. A method forproducing an EB virus vector without viral replication ability,comprising inducing lytic infection of an Akata packaging cellintroduced with amplicon plasmid, which is produced by the method ofclaim 4, thereby allowing an EB virus vector covered with a virusenvelope to be released.
 6. A method for producing an immortalized Blymphocyte, comprising inducing lytic infection of an Akata packagingcell introduced with amplicon plasmid, which is produced by the methodof claim 4, thereby allowing an EB virus vector covered with a virusenvelope to be released, and infecting a B lymphocyte with the resultingEB virus vector.